The mouse inner cell mass (ICM) and the embryonic stem (ES) cells derived from it contain two active X chromosomes. Similarly, nuclear reprogramming resets the state of X chromosome inactivation (XCI) in mouse induced pluripotent stem (IPS) cells, which also transcribe both of their X chromosomes. However, the proper status of dosage compensation in human pluripotent stem cells remains to be clarified. As the transcriptional networks that propagates the pluripotent state in mouse ES cells has been tightly linked to the regulation of X chromosome inactivation, it will be of substantial interest to determine whether these gene regulatory processes are also tightly coupled within human pluripotent stem cells. In addition, many diseases result from mutations in X-linked genes. As there is substantial interest in using reprogrammed cells for modeling these conditions in vitro, it will be critically important to understand the state of dosage compensation in both human IPS cells and their differentiated derivatives. Here we propose to combine reprogramming, stem cell and genomic approaches to understand the behavior of the inactive X chromosome during the generation, maintenance and differentiation of human IPS cells. Our specific aims are to: Aim 1) To determine whether female IPS cells inherit the inactive X chromosome of the somatic cells from which they are derived and to determine how stably they maintain this inactive X in the course of long-term culture. Aim 2) To determine whether the loss of cytological hallmarks of X chromosome inactivation is accompanied by X-chromosome-wide relaxation of DNA methylation, chromatin structure and transcriptional silencing. Aim 3) We will determine the specific culture conditions that contribute to instability of X chromosome inactivation that we have observed and identify culture conditions that allow proper maintenance of X chromosome-wide heterochromatin.